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1
Leavitt, Markley Carl. "Bacteriophage T5 DNA polymerase relationships of DNA polymerases." Diss., The University of Arizona, 1990. http://hdl.handle.net/10150/185335.
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T5 DNA polymerase, a highly processive single polypeptide enzyme, and PRD1 DNA polymerase, a protein-primed DNA polymerase, have been analyzed for their primary structural features. The amino acid sequence of T5 DNA polymerase reveals a high degree of homology with DNA polymerase I (Pol I) of Escherichia coli and retains many of the amino acid residues which have been implicated in the 3'-5' exonuclease and DNA polymerase activities of that enzyme. Alignment with sequences of polymerase I and T7 DNA polymerase (family A polymerases) was used to identify regions possibly involved in the high processivity of this enzyme. Further amino acid sequence comparisons of T5 DNA polymerase with a large group of DNA polymerases (family B) previously shown to exhibit little similarity to Pol I, indicate certain sequence segments are shared among distantly related DNA polymerases. These shared regions have been implicated in the 3'-5' exonuclease function of Pol I which suggests that the proofreading domains of all these enzymes may be related. Mutations in these segments in T5 DNA polymerase (family A) and PRD1 DNA polymerase (family B) greatly decrease the exonuclease activity of these enzymes but leave the polymerase activities intact. Additionally, an exonuclease deficient T5 DNA polymerase is used in DNA sequencing reactions and yields consistent results with low background contamination on autoradiographs of polyacrylamide/urea gels. PRD1 mutants defective in 3 regions which are highly conserved among family B DNA polymerases, are deficient in DNA polymerase activity but retain exonuclease activity.
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Roettger, Michelle P. "Insight into the Fidelity of Two X-Family Polymerases: DNA Polymerase Mu and DNA Polymerase Beta." Columbus, Ohio : Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1211074588.
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SALHI, SAMIA. "Dna polymerase de sulfolobus acidocaldarius : interet de l'etude des dna polymerases thermophiles." Paris 7, 1989. http://www.theses.fr/1989PA077169.
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La copie par la dna polymerase de sulfolobus acidocaldarius d'un dna simple brin uni-amorce de sequence connue a ete etudiee. Les parametres cinetiques affectant cette synthese dependent de la sequence du dna. La temperature de reaction a son importance. Les methodes employees sont la reaction polymerase en chaine, la mutagenese dirigee, le sequencage de sanger
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Pospiech, H. (Helmut). "The role of DNA polymerases, in particular DNA polymerase ε in DNA repair and replication." Doctoral thesis, University of Oulu, 2002. http://urn.fi/urn:isbn:9514266692.
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Abstract
Analysis of the primary structure of DNA polymerase ε B subunit
defined similarities to B subunits of eukaryotic DNA polymerases α,
δ and ε as well as the small subunits of DNA polymerase DI of
Euryarchaeota. Multiple sequence alignment of these proteins revealed the
presence of 12 conserved motifs and defined a novel protein superfamily.
The members of the B subunit family share a common domain architecture,
suggesting a similar fold, and arguing for a conserved function among
these proteins.
The contribution of human DNA polymerase ε to nuclear DNA
replication was studied using the antibody K18 that specifically inhibits
the activity of this enzyme in vitro. This antibody
significantly inhibited DNA synthesis both when microinjected into nuclei
of exponentially growing human fibroblasts and in isolated HeLa cell
nuclei, but did not inhibit SV40 DNA replication in
vitro. These results suggest that the human DNA polymerase
ε contributes substantially to the replicative synthesis of DNA and
emphasises the differences between cellular replication and viral model
systems.
The human DNA polymerases ε and δ were found capable of
gap-filling DNA synthesis during nucleotide excision repair in
vitro. Both enzymes required PCNA and the clamp loader RFC, and
in addition, polymerase δ required Fen-1 to prevent excessive
displacement synthesis. Nucleotide excision repair of a defined DNA lesion
was completely reconstituted utilising largely recombinant proteins, only
ligase I and DNA polymerases δ and ε provided as highly purified
human enzymes. This system was also utilised to study the role of the
transcription factor II H during repair.
Human non-homologous end joining of model substrates with different
DNA end configurations was studied in HeLa cell extracts. This process
depended partially on DNA synthesis as an aphidicolin-dependent DNA
polymerase was required for the formation of a subset of end joining
products. Experiments with neutralising antibodies reveal that DNA
polymerase α but not DNA polymerases β or ε, may represent
this DNA polymerase activity. Our results indicate that DNA synthesis
contributes to the stability of DNA ends, and influences both the
efficiency and outcome of the end joining event. Furthermore, our results
suggest a minor role of PCNA in non-homologous end joining.
5
Mischo, Hannah. "Disengaging Polymerases : Transcriptional termination by RNA polymerase II in Saccharomyces cerevisiae and the maintenance of genome integrity." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.514968.
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Efthimiopoulos, Georgia. "Bypass of N2-Deoxyguanosinyl Adducts by DNA Polymerases and Kinetic Implications for Polymerase Switching." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1366297865.
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Siot, Alexandra. "Elaboration et caractérisation de polymères nanochargés." Thesis, IMT Mines Alès, 2018. http://www.theses.fr/2018EMAL0001.
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Linley, M. "The detection of polymerase inhibiting lesions using the polymerase arrest polymerase chain reaction assay." Thesis, Swansea University, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.637924.
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There is a constant need to determine the genotoxic potential of the agents to which the human population is exposed. The stringent testing of new products is legislatively controlled and dependent on the accumulation of sufficient scientific data to allow an analysis of the risk. It is important to predetermine any risks in the workplace prior to the presentation of disease and to provide factual public information on personal exposure e.g. the risks associated with UV light. Various experimental assays have been developed to assess the genotoxicity, mutagenicity and mcarcinogenicity of given physical and chemical agents. The Polymerase Arrest- Polymerase Chain Reaction (PA-PCR) assay was employed to investigate the genotoxic effects (DNA adducts, DAN strand breaks and DNA crosslinking) of various physical and chemical agents on naked isolated DNA. The assay was modified to provide two adapted methods, which increased the sensitivity of the assay to report DNA damage at significantly lowered exposure levels. The ability of the PA-PCR assay to perform as an initial screening process for genotoxic activity was assessed and determined.
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Cramer, Janina. "Funktionelle Charakterisierung der RNA-abhängigen RNA-Polymerase des Hepatitis-C-Virus Untersuchung molekularer Mechanismen der Substratspezifität von DNA-abhängigen DNA-Polymerasen /." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971700796.
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Sikorsky, Jan A. "Effect of DNA base modification on polymerase chain reaction efficiency and fidelity." Huntington, WV : [Marshall University Libraries], 2005. http://www.marshall.edu/etd/descript.asp?ref=554.
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Lawrence, Michael S. (Michael Scott) 1975. "[RNA polymerase ribozymes]." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/31193.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Title supplied by cataloger from abstract.
Includes bibliographical references.
The RNA World is a hypothetical ancient evolutionary era during which RNA was both genome and catalyst. During that time, RNA was the only kind of enzyme yet in existence, and one of its chief duties was the replication of RNA. This scenario presupposes that among all possible RNA sequences, there exist RNA replicase ribozymes, capable of synthesizing RNA using the information in an RNA template. The goal of the present work is to provide experimental evidence in support of this conjecture, by isolating such ribozymes in the laboratory. We created a large pool of RNA molecules each containing a previously isolated RNA ligase ribozyme and a large stretch of random RNA. Applying in vitro evolution to select for molecules that could extend a tethered RNA primer using nucleoside triphosphates, we isolated nine distinct classes of polymerase ribozymes. Two of these rudimentary polymerases were further evolved to the point that they each could add 14 nucleotides to an untethered primer-template. One of them was subjected to a detailed further characterization. The polymerization it catalyzes was shown to be accurate, with an average fidelity of nearly 97%. It was shown to be general, with primer-templates of all sequences and lengths being accepted as substrates. Finally, it was shown to be partially processive, with the polymerase achieving processivity as high as 90% in a few instances. The polymerase is currently limited by its low affinity for the primer-template. Future work will focus on improving primer- template binding, in order to produce a polymerase that can synthesize longer RNA.
Michael S. Lawrence.
Ph.D.
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Armache, Karim. "Crystal structures of the complete 12-subunit RNA polymerase II and its subcomplex Rpb4/7, and modeling of RNA polymerases I and III." Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-49156.
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Armache, Karim-Jean. "Crystal structures of the complete 12-subunit RNA polymerase II and its subcomplex Rpb4-7, and modeling of RNA polymerases I and III." [S.l.] : [s.n.], 2005. http://edoc.ub.uni-muenchen.de/archive/00004915.
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Folkesson, Carl, and Ola Christensson. "Genotypning av laktostolerans (LCT-13910C>T) direkt på blod med realtids-PCR : Utvärdering av Kapa Probe Force." Thesis, Hälsohögskolan, Högskolan i Jönköping, HHJ, Avd. för naturvetenskap och biomedicin, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:hj:diva-30807.
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Hos vuxna individer förekommer två fenotyper gällande produktionen av laktas, vilka kallas laktostolerans och laktosintolerans. Vid laktosintolerans produceras otillräckliga mängder laktas vilket framkallar symptom som magsmärtor och flatulens vid intagandet av mjölkprodukter. En enbaspolymorfism (LCT-13910C>T) har kopplats till laktostolerans hos nordvästeuropéer och kan genotypas med smältkurveanalys i realtids-PCR. På Laboratoriemedicin vid Länssjukhuset Ryhov används idag en metod vid genotypning av LCT-13910C>T där extraktion av DNA från blod krävs innan analys. Anledningen till detta är att DNA-polymeraset som ingår enzymmixen LightCycler® FastStart DNA Master HybProbe endast fungerar med rent DNA-templat. Med en annan enzymmix, Kapa Probe Force, ska analys kunna göras direkt på blod. För att utvärdera enzymmixen jämfördes resultat från befintlig metod och resultat från metod med Kapa Probe Force, gällande förmågan att identifiera genotyperna LCT-13910C/C, C/T och T/T samt med avseende på imprecision. Vid jämförelse mellan metoderna samstämde resultatet i avseende på genotyp till 100 % utifrån specificerade smälttemperaturer (Tm) för respektive genotyp angivna i kitet för primer/prober. Däremot syntes lägre fluorescensnivå på smältopparna i metod med Kapa Probe Force, men påverkade inte tolkning av smältkurvorna. En lägre prov-till-prov-variation sågs även i resultatet från metod med Kapa Probe Force gentemot befintlig metod.Among adults two phenotypes are found with regards to production of lactase, these are termed lactase persistence and lactose intolerance. Lactose intolerance is characterized by a low production of lactase, which leads to symptoms such as stomach ache and flatulence after the consumption of dairy products. A single nucleotide polymorphism (LCT-13910C>T) has been correlated with the occurrence of lactase persistence in northwestern Europeans. Genotyping of LCT-13910C>T is possible with melting curve analysis in real time PCR. The currently used method for genotyping of LCT-13910C>T at Ryhov County Hospital requires the extraction of DNA template from blood, due to the fact that the DNA-polymerase in the kit LightCycler® FastStart DNA Master HybProbe requires pure DNA template for analysis. With another DNA-polymerase, included in the kit Kapa Probe Force, analysis on crude samples such as pure blood should be possible. Evaluation of Kapa Probe Force included comparison of the results from both methods with regards to identification of genotypes LCT-13910C/C, C/T and T/T and with regard to imprecision. The results from Kapa Probe Force were 100 % consistent with the results from existing method and acquired melting temperatures (Tm) were all within the accepted ranges specified in the kit of primers and probes. The fluorescence of melting curves acquired with Kapa Probe Force was significantly lower, however this had no effect when it came to interpreting the results. A lower variation could also be seen between samples with Kapa Probe Force compared to existing method.
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Sun, Hui. "Enhancing analytical capability of piezoelectric quartz crystal and capillary electrophoresis in environmental analysis using polymerase chain reaction, molecularly imprinted polymers and nanotechnology." Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36959522.
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Chan, Annie Yee-Man. "Interactions between the influenza virus RNA polymerase and cellular RNA polymerase II." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670083.
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Neugebauer, Karla M., Inna Grishina, Anita S. Bledau, and Imke Listerman. "Extragenic Accumulation of RNA Polymerase II Enhances Transcription by RNA Polymerase III." PLOS, 2007. https://tud.qucosa.de/id/qucosa%3A27951.
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Recent genomic data indicate that RNA polymerase II (Pol II) function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III). Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP) and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated ∼300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was α-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon α-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome.
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Neugebauer, Karla M., Inna Grishina, Anita S. Bledau, and Imke Listerman. "Extragenic Accumulation of RNA Polymerase II Enhances Transcription by RNA Polymerase III." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-184076.
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Recent genomic data indicate that RNA polymerase II (Pol II) function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III). Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP) and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated ∼300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was α-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon α-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome.
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Lee, Sally. "Architecture of RNA polymerase II and RNA polymerase III pre-initiation transcription complexes /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/9213.
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Moussa, Sougueh Charmarke. "Elaboration et étude de films de polypyrroles et de polycarbazoles fonctionnalisés obtenus par oxydation électrochimique." Thesis, Bourgogne Franche-Comté, 2019. http://www.theses.fr/2019UBFCD024.
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Description détaillée du sujet : Au cours des dernières années, les électrodes recouvertes d’un film polymère déposé par voie électrochimique ont suscité un engouement croissant dans le domaine des capteurs chimiques et des biocapteurs. Un exemple d’application est celui des capteurs de pH potentiométriques qui utilisent les atomes d’azote présents dans des polymères pour détecter des variations de pH. Ces électrodes sont intéressantes en vue d’applications cliniques et biologiques comme l’analyse in vivo dans la mesure où elles sont, contrairement aux électrodes de pH traditionnelles, miniaturisables tout en étant biocompatibles si les polymères choisis le sont également. Le développement d’un capteur enzymatique permettant de quantifier la présence d’urée dans un échantillon liquide est également d’un intérêt significatif dans la mesure où l’urée, qui est présente dans l’organisme humain, est un des indicateurs biologiques du bon fonctionnement des reins. En effet, des taux élevés d’urée révèlent une insuffisance rénale. De plus le contrôle des quantités d’urée est d’intérêt dans l’industrie agro-alimentaire et le contrôle environnemental. Les polymères électrodéposés peuvent également être utilisés comme supports de culture de cellules biologiques ce qui peut permettre de développer des applications dans le domaine de la médecine, par exemple pour le développement de prothèses, la lutte contre les maladies neurogénératrices, la création de muscles artificiels. Par conséquent, nous souhaitons proposer une thèse portant sur l’élaboration de films polymères originaux permettant le développement de nouveaux (bio)capteurs électrochimiques et de promouvoir des applications dans le domaine de la médecineDetailed description of the subject: during the last years, electrodes covered with a polymer movie put down by electrochemical way aroused a craze growing in the field of the chemical sensors and the biosensors. An example of application is the one sensors of pH potentiométriques who use the present atoms of nitrogen in polymers to detect variations of pH. These electrodes are interesting with the aim of applications clinical and biological as the in vivo analysis as far as they are, contrary to the traditional electrodes of pH, miniaturisables while being biocompatible if the chosen polymers are also. The development of an enzymatic sensor allowing to quantify the presence of urea in a liquid sample is also
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Chang, Ya-Wen. "The Ras/PKA pathway controls transcription of genes involved in stationary phase entry in Saccharomyces cerevisiae." Columbus, Ohio : Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1061214472.
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Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xiii, 108 p.; also includes graphics. Includes abstract and vita. Advisor: Paul K. Herman, Dept.of Molecular, Cellular, and Developmental Biology. Includes bibliographical references (p. 96-108).
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Wolnik, Anna. "Association des copolymères à séquences (1->4)-a-L-guluronane en présence d’ions calcium." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV003/document.
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Les alginates forment des gels transparents en solution aqueuse en présence de certains ions divalents. Cette propriété est principalement attribuée à la formation de zones de jonction impliquant les séquences (1->4)-a-L-guluronane de chaînes adjacentes. Des oligomères d'alginates ont été utilisés comme briques élémentaires pour la synthèse de polymères biohybrides contenant des chaines pendantes oligo-(1->4)-a-L-guluronane. La rhéologie et la diffusion de la lumière ont permis d'étudier leur gélification ionotropique. De plus, une image atomistique des associations entre chaines latérales a été donnée grâce à la modélisation moléculaire et la microscopie de force atomique. Les polymères biohybrides portant des résidus pendant (1->4)-a-L-guluronane forment des gels en présence de Ca2+. L'addition de blocs guluronane ou mannuronane au gel préformé fait diminuer sa force avec quasiment la même efficacité. L'étude par dynamique moléculaire de séquences (1->4)-a-L-guluronan totalement chargées en présence d'ions Ca2+ suggère qu'environ 8 unités de répétition sont suffisantes pour former spontanément des zones de jonction. De plus, l'analyse conformationnelle de duplexes de chaines (1->4)-a-L-guluronane ayant 12 unités de répétition révèle une grande variété de conformations accessibles, ce qui est consistant avec la difficulté d'obtention de cristaux de Ca2+-guluronate de dimension suffisante pour les études cristallographiques. Les forces d'adhésion entre des homo-oligomères d'alginates en présence de Ca2+ mesuré par spectroscopie de force atomique montrent que la force d'interaction croit selon l'ordre suivant: M-M < M-G or G-M < G-G. Un résultat important est que les blocs mannuronanes, en complexe avec le calcium, peuvent être impliqués dans des associations homotypiques et hétérotypiques. Ce résultat est consistant avec la détection d'agrégats d'oligomères de mannuronanes observés en diffusion de la lumière pendant l'addition de CaCl2. Les blocs M contribuent donc également à la formation du gel mais la force associée est plus faible que celle des blocs GAlginates form transparent hydrogels in aqueous solution upon addition of some divalent cations. This property is mostly due to the formation of junction zones involving (1->4)-a-L-guluronan sequences on adjacent polymer chains. Oligomers of alginates were used as molecular bricks for the synthesis of biohybrid polymers featuring (1->4)-a-L-guluronan sequences as side chains. Rheology and Light Scattering have been applied to investigate their ionotropic gelation. In addition, an atomistic picture of the Ca2+-driven side chain associations was also provided thanks to Molecular Modeling and Atomic Force Spectroscopy. Biohybrid polymers carrying (1->4)-a-L-guluronan residues formed soft and transparent hydrogels in the presence of Ca2+. The addition of either guluronan, or mannuronan blocks to the pre-formed gel reduced its strength almost with the same efficiency. A molecular dynamics investigation of fully charged (1->4)-a-L-guluronan sequences in the presence of a neutralizing amount of Ca2+ ions suggested that about 8 repeating units may be sufficient to the spontaneous formation of junction zones. Furthermore, conformational analysis of (1->4)-a-L-guluronan chain having 12 repeating units in duplexes revealed a wide variety of accessible conformations, a feature consistent with the general difficulty in obtaining crystals of Ca2+-guluronate with suitable lateral dimensions for crystallographic studies. The adhesion forces between homo-alginate oligomers in the presence of Ca2+ measured by Atomic Force Spectroscopy showed that the strength of interactions increased in the following order: M-M < M-G or G-M < G-G. One of the most significant findings to emerge from this study is that mannuronan blocks complexed via calcium ions can be involved in both homotypic and heterotypic associations. This result is consistent with the detection of aggregates observed for mannuronan oligomers by Light Scattering during the addition of CaCl2. Thus, M-blocks also contribute to the gel formation but their strength seemed to be however weaker than G-blocks
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Showalter, Alexander Keith. "KINETIC STUDIES OF TWO ERROR-PRONE DNA REPAIR ENZYMES: POSSIBLE MECHANISMS FOR VIRAL MUTAGENESIS." Connect to this title online, 2002. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1016207119.
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Thesis (Ph. D.)--Ohio State University, 2002.Title from first page of PDF file. Document formatted into pages; contains xii, 97 p.; also contains graphics (some col.). Includes abstract and vita. Advisor: Ming-Daw Tsai, Dept. of Chemistry. Includes bibliographical references (p. 92-97).
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Burrows, Judith Ann. "DNA polymerase from Bacillus caldotenax." Thesis, Open University, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359180.
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Niedbala, Angela Rochelle. "Kinetic studies of transcription initiation by wild type T7 RNA polymerase, his-tagged wild type T7 RNA polymerase and GP1-Lys222 T7 RNA polymerase." Thesis, Georgia Institute of Technology, 1995. http://hdl.handle.net/1853/27288.
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Sutcliffe, Josephine E. "The regulation of RNA polymerase I and RNA polymerase III transcription by the pocket proteins." Thesis, University of Glasgow, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327577.
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Zhu, Weiguo. "Structure-function analysis of DNA polymerase: Purification, characterization and in vitro mutagenesis of PRD1 DNA polymerase." Diss., The University of Arizona, 1993. http://hdl.handle.net/10150/186573.
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A small lipid-containing bacteriophage PRD1 encodes a DNA polymerase that utilizes a protein primer for the initiation of DNA replication. The purification of PRD1 DNA polymerase has been hampered by the insolubility of the overexpressed enzyme in E. coli cells. A simple and rapid procedure for purification of the overexpressed PRD1 DNA polymerase has been developed. This method is based on guanidine hydrochloride denaturation and renaturation of the insoluble PRD1 DNA polymerase overexpressed in E. coli containing recombinant plasmid pEJG. The purified DNA polymerase was extensively characterized and found to be indistinguishable from the normal soluble PRD1 DNA polymerase as judged by enzymatic properties. These properties include: protein-primed initiation of PRD1 DNA replication, strand-displacement DNA synthesis, DNA polymerase processivity, 3' to 5' exonuclease activity and filling-in repair type DNA synthesis. Furthermore, the kinetic parameters determined for dNTPs and primer-terminus were of the same order of magnitude. The availability of a simple purification procedure for PRD1 DNA polymerase should permit detailed structure-function analysis of this enzyme. All known family B DNA polymerases contain a conserved region of amino acids, KX₆₋₇YG, which appears to be correspond to the "finger" alpha helix O of the Klenow fragment of E. coli DNA polymerase I, a family A DNA polymerase. A site-directed mutagenesis study has been applied to access the functional role of the invariant amino acid lysine-340 of PRD1 DNA polymerase. Mutant DNA polymerases were overexpressed and purified to near homogeneity. The results showed that the modification of the lysine-340 of PRD1 DNA polymerase abolishes the polymerase activity without affecting the 3' to 5' exonuclease activity. Site-directed mutagenesis studies revealed that residues important for the 3' to 5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in PRD1 DNA polymerase as well. Although PRD1 DNA polymerase has a smaller 3' to 5' exonuclease domain, active sites appear to be very similar to those of the Klenow fragment. Moreover, the metal binding ligands were also found to be important for the strand-displacement activity, a unique feature of PRD1 DNA polymerase.
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Swiatecka-Hagenbruch, Monika. "Phagenähnliche RNA-Polymerasen." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15924.
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Chloroplasten höherer Pflanzen haben kleine Genome. Trotzdem ist ihre Transkriptionsmaschinerie sehr komplex. Plastidäre Gene werden von plastidenkodierten (PEP) und kernkodierten RNA-Polymerasen (NEP) transkribiert. In der vorliegenden Arbeit wurden Promotoren plastidärer Gene und Operons von Arabidopsis thaliana charakterisiert. Zur Unterscheidung zwischen NEP- und PEP-Promotoren wurden erstmals spectinomycinbehandelte, chlorophylldefiziente Arabidopsis-Pflanzen mit fehlender PEP-Aktivität verwendet. Obwohl für einige Gene auch einzelne Promotoren lokalisiert wurden, wird die Transkription der meisten plastidären Gene und Operons an multiplen Promotoren initiiert. Der Vergleich plastidärer Promotoren von Tabak und Arabidopsis zeigte eine hohe Vielfältigkeit der Promotornutzung, die möglicherweise auch in anderen höheren Pflanzen vorkommt. Dabei stellt die individuelle Promotornutzung eine speziesspezifische Kontrollmöglichkeit der plastidären Genexpression dar. Das Kerngenom von Arabidopsis beinhaltet zwei Kandidatengene der NEP, RpoTp und RpoTmp, welche Phagentyp-RNA-Polymerasen kodieren. In der vorliegenden Arbeit wurde die Wirkung veränderter RpoTp-Aktivität auf die Nutzung von NEP- und PEP-Promotoren in transgenen Arabidopsis-Pflanzen mit verminderter und fehlender RpoTp-Aktivität untersucht. Im Keimlingsstadium konnten Unterschiede in der Promotornutzung zwischen Wildtyp und Mutanten beobachtet werden. Fast alle NEP-Promotoren wurden in Pflanzen mit verringerter oder fehlender RpoTp-Aktivität genutzt. Dabei zeigten nur einige von ihnen eine geringere Aktivität, andere wiederum waren sogar verstärkt aktiv. Der starke NEP-Promotor des essentiellen ycf1 Gens wurde in jungen Keimlingen ohne funktionelle RpoTp nicht genutzt. Die Ergebnisse zeigen, dass NEP gemeinsam von beiden Phagentyp-RNA-Polymerasen RpoTp und RpoTmp repräsentiert wird und dass beide sowohl eine überlappende, als auch eine spezifische Rolle in der Transkription plastidärer Gene innehaben.Although chloroplasts of higher plants have small genomes, their transcription machinery is very complex. Plastid genes of higher plants are transcribed by the plastid-encoded plastid RNA polymerase PEP and the nuclear-encoded plastid RNA polymerases NEP. Here, promoters of plastid genes and operons have been characterized in Arabidopsis thaliana. For the first time spectinomycin-treated, chlorophyll-deficient Arabidopsis plants lacking PEP activity have been used to discriminate between NEP and PEP promoters. Although there are plastid genes that are transcribed from a single promoter, the transcription of plastid genes and operons by multiple promoters seems to be a common feature. Comparison of plastid promoters from tobacco and Arabidopsis revealed a high diversity, which my also apply to other plants. The diversity in individual promoter usage in different plants suggests that there are species-specific solutions for attaining control over gene expression in plastids. The nuclear genome of Arabidopsis contains two candidate genes for NEP transcription activity, RpoTp and RpoTmp, both coding for phage-type RNA polymerases. In this study the usage of NEP and PEP promoters has been analysed in transgenic Arabidopsis plants with reduced and lacking RpoTp activity. Differences in promoter usage between wild type and mutant plants were most obvious early in development. Nearly all NEP promoters were active in plants with low or lacking RpoTp activity, though certain promoters showed reduced or even increased usage. The strong NEP promoter of the essential ycf1 gene was not transcribed in young seedlings without functional RpoTp. These results provide evidence for NEP being represented by two phage-type RNA polymerases RpoTp and RpoTmp that have overlapping as well as specific functions in the transcription of plastid genes.
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Chadima, David. "Technologie emulzní polymerace." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2016. http://www.nusl.cz/ntk/nusl-240786.
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The diploma thesis deals with the influence of technological parameters on the emulsion copolymerization of methyl methacrylate with n butyl acrylate. The theoretical part contains knowledge in the field of influences of proces tempereture, concentration of initiator, concentration of emulsifier, stirring rate and dose rate on emulsion polymerization. In the experimental part was observed effect of the concentration of the ionic emulsifier, nonionic emulsifier, furthemore was observed effect of stirring speed and the concentration of initiator K2S2O8 on conversion of copolymerization. During all copolymerizations, conversions was determinated via solids content evaluation. By dynamic light scattering was determinated and presented polymer particle size of the prepared emulsions. Stability of emuldions was observed via effect of different ionic strenght and yield of freeze thaw cycles coalescence. Based on experimental results were proposed conditions of polyacrylic lattex preparation.
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Kuhn, Claus-Dieter. "Functional Architecture of RNA polymerase I." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-81136.
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Buttner, M. "RNA polymerase - DNA interactions in Streptomyces." Thesis, University of Bristol, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.354445.
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Patel, Premal Harshad. "Evolution of DNA polymerase active site /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/6361.
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Ozdemir, Ahmet Yunus. "BIOCHEMICAL STUDIES OF DNA POLYMERASE THETA." Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/560412.
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Biomedical SciencesPh.D.
POLQ is a unique multifunctional replication and repair gene that encodes a multidomain protein with a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase θ-helicase (Polθ-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Polθ-helicase (residues 1– 894) efficiently unwinds DNA with 3'–5' polarity, including DNA with 3' or 5' overhangs, blunt- ended DNA, and replication forks. Polθ-helicase also efficiently unwinds RNA-DNA hybrids and exhibits a preference for unwinding the lagging strand at replication forks, similar to related HELQ helicase. Finally, we find that Polθ-helicase can facilitate strand displacement synthesis by Polθ-polymerase, suggesting a plausible function for the helicase domain. Taken together, these findings indicate nucleic acid unwinding as a relevant activity for Pol theta in replication repair. DNA polymerase theta is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining of DNA double-strand breaks. How full-length human DNA polymerase theta performs microhomology-mediated end-joining and is regulated by the helicase and disordered central domain remains unknown. We find that the helicase upregulates DNA polymerase theta microhomology-mediated end-joining activity in an ATPase-independent manner. Using single-particle microscopy, we find that DNA polymerase theta forms large multimeric complexes that promote DNA accumulation and end-joining. We further find that the disordered central domain regulates DNA polymerase theta multimerization and governs its DNA substrate requirements for end-joining. In summary, these studies identify major regulatory functions for the helicase and central domains in DNA end-joining and the structural organization of DNA polymerase theta.
Temple University--Theses
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Che, Austin 1979. "Fluorescence assay for polymerase arrival rates." Thesis, Massachusetts Institute of Technology, 2003. http://hdl.handle.net/1721.1/16618.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, February 2004.Includes bibliographical references (p. 87-100).
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
To engineer complex synthetic biological systems will require modular design, assembly, and characterization strategies. The RNA polymerase arrival rate (PAR) is defined to be the rate that RNA polymerases arrive at a specified location on the DNA. Designing and characterizing biological modules in terms of RNA polymerase arrival rates provides for many advantages in the construction and modeling of biological systems. PARMESAN is an in vitro method for measuring polymerase arrival rates using pyrrolo-dC, a fluorescent DNA base that can substitute for cytosine. Pyrrolo-dC shows a detectable fluorescence difference when in single-stranded versus double-stranded DNA. During transcription, RNA polymerase separates the two strands of DNA, leading to a change in the fluorescence of pyrrolo-dC. By incorporating pyrrolo-dC at specific locations in the DNA, fluorescence changes can be taken as a direct measurement of the polymerase arrival rate.
by Austin Che.
S.M.
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Feugeas, Olivier. "Pcr (polymerase chain reaction) et vih." Lille 2, 1990. http://www.theses.fr/1990LIL2M264.
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Smith, Brian A. "Mechanistic insights into a reverse polymerase." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1353507187.
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White, Eleanor. "Transcription termination by RNA polymerase II." Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.558432.
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RNA Polymerase II (Pol I1) is responsible for the transcription of all protein-encoding genes. Pol II termination is dependent on RNA processing signals (both terminal intron splice sites, and cleavage and polyadenylation signals) as well as specific terminator elements located downstream of the poly(A) site. Detailed analysis of the human ~- globin gene terminator has shown that it contains a sequence-specific region that promotes rapid Co-Transcriptional Cleavage (CoTC) of the nascent transcript - an essential but not well understood step in the human ~-globin gene termination process. In the first part of this thesis, the role of sequences within this CoTC-mediated terminator element in the termination process is investigated. Analysis of mutant terminator sequences indicate that homopolymer A tracts are important for Pol II termination. The second part of this study focuses on identifying the activity responsible for CoTC, by using the yeast S. pombe as a tool for genetic analysis. The results indicate that the human ~-globin gene terminator is inefficient in S. pombe, suggesting that a mammalian specific factor(s) are required. In the final part of this study, I describe an investigation into the possibility that the exosome subunit Dis3 or the RNase III enzyme Dicer are involved in CoTC mediated transcription termination. While Dis3 is not involved in the CoTC process my results on Dicer may imply a significant role. Lastly, I present a preliminary investigation into the effect of pre-mRNA processing and the carboxyl terminal domain (CTD) of Po 1 II on CoTC activity.
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Jokela, M. (Maarit). "Replicative DNA polymerase associated B-subunits." Doctoral thesis, University of Oulu, 2004. http://urn.fi/urn:isbn:9514274814.
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Abstract
Replicative DNA polymerases (pols) synthesize chromosomal DNA with high accuracy and speed during cell division. In eukaryotes the process involves three family B pols (α, δ, ε), whereas in Archaea, two types of pols, families B and D, are involved. In this study the B-subunits of replicative pols were analysed at the DNA, RNA and protein levels.
By cloning the cDNAs for the B-subunits of human and mouse pol ε we were able to show that the encoded proteins are not only homologous to budding yeast pol ε, but also to the second largest subunit of pol α. Later studies have revealed that the B-subunits are conserved from Archaea to human, and also that they belong to the large calcineurin-like phosphoesterase superfamily consisting of a wide variety of hydrolases.
At the mRNA level, the expression of the human pol ε B-subunit was strongly dependent on cell proliferation as has been observed for the A-subunit of pol ε and also for other eukaryotic replicative pols. By analysing the promoter of the POLE2 gene encoding the human pol ε B-subunit we show that the gene is regulated by two E2F-pocket protein complexes associated with the Sp1 and NF-1 transcription factors. Comparison of the promoters of the human pol ε and the pol α B-subunit indicates that the genes for the B-subunits may be generally regulated through E2F-complexes whereas adjustment of the basal activity may be achieved by distinct transcription factors.
To clarify the function of the B-subunits, we screened through the expression of 13 different recombinant B-subunits. Although they were mainly expressed as insoluble proteins in E. coli, we were able to optimize the expression and purification for the B-subunit (DP1) of Methanococcus jannaschii pol D (MjaDP1). We show that MjaDP1 alone was a manganese dependent 3'-5' exonuclease with a preference for mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreader of archaeal pol D. So far, pol D is the only pol family utilising an enzyme of the calcineurin-like phosphoesterase superfamily as a proofreader.
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Tran, Tuan Anh. "Screening against the dengue virus polymerase." Thesis, Aix-Marseille, 2016. http://www.theses.fr/2016AIXM4006.
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La dengue, une des maladies les plus largement émergents actuellement, avec 390 millions d'infections chaque année (OMS), est causée par le virus de la dengue contre lequel il n’existe pas de traitements. La protéine NS5 a un rôle important dans le cycle de réplication. Cette protéine se compose d'une méthionine S-transférase d’adénosyl en N-terminal et une ARN polymérase dépendante de l'ARN (RdRp) en C-terminal. Cette NS5 RdRp peut catalyser non seulement la synthèse du brin négatif de l'ARN, utilisé comme matrice pour synthétiser l'ARN brin plus-supplémentaire, mais aussi pour la synthèse d'un ARN complémentaire à partir d'une matrice court e d'ARN sans amorce (de novo). Dans ce travail de thèse, nous présentons la production et le test de l'activité de la protéine NS5, ainsi que du domaine polymérase RdRp pour les quatre sérotypes du virus de la dengue en développant un nouveau test enzymatique, en utilisant comme un réactif fluorescent. L'utilisation de ce réactif fluorescent a également contribué à la détermination des conditions optimisées pour développer un essai de criblage de l'activité polymérase pour identifier des inhibiteurs contre le virus de la dengue. En outre, quatre flavonoïdes, Hinokiflavone, apigénine, la quercétine et Amentoflavone ont montré des valeurs d’IC50 équivalentes contre toutes les constructions NS5 et les domaines polymérase des quatre sérotypesDengue fever, one of the most widely emerging diseases nowadays with 390 million infections each year (WHO), is caused by Dengue virus in which no official antiviral reagent or vaccine is available. The NS5 protein has an important role in the replication cycle. This protein consists of a S-adenosyl methionine transferase at N-terminal and a RNA dependent RNA polymerase (RdRp) at C-terminal. This NS5 RdRp can catalyse for not only synthesis of minus-strand RNA to be used as the template to synthesize additional plus-strand RNA but also synthesizing a complement RNA from a short RNA template without primer (de novo). In this research we present the production and activity test for NS5 protein and N-terminal extended sequence 266-900 from NS5 RdRp of all first four serotypes of Dengue virus and a construct of sequence 273-900 using a new enzymatic assay, using Picogreen as fluorescent reagent. Using this fluorescent reagent also helped determining the optimised conditions to develop a screening assay for inhibitors against dengue polymerase activity. In addition, four flavonoids, Hinokiflavone, Apigenin, Quercetin and Amentoflavone showed approximate IC50 values when testing on all NS5 and polymerase protein constructs of all four serotypes
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Ringel, Eva Rieke. "Molecular basis of RNA polymerase III transcription repression by Maf1 & Structure of human mitochondrial RNA polymerase." Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-134070.
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Chin, Wing-hong, and 錢永康. "Polymerase activity of chimeric polymerase : a determining factor for an influenza virus to be a pandemic strain." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hdl.handle.net/10722/193398.
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The influenza polymerase is a complex of three subunits, polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1) and polymerase acidic protein (PA). It associates with the viral RNA segment and nucleoprotein (NP) to form a viral ribonucleoprotein (vRNP) complex which is important for transcription and replication of the viral genome. Concurrently, the previous three influenza pandemics viruses contain reassorted vRNP of different origins. This leads to the aim of study to investigate the role of polymerase in the pandemic viruses. By reconstitution of vRNPs in human cells, it was demonstrated that vRNPs of H2N2 and H3N2 pandemic viruses had higher polymerase activity than the H2N2 seasonal viruses in-between them. The recombinant virus with H2N2 pandemic vRNP also showed faster growth kinetics in the early stage of viral replication and better adaptability to the selective environment with neuraminidase inhibitor than the recombinant virus with H2N2 seasonal vRNP, which had a lower polymerase activity. Reconstitution of chimeric vRNPs of H2N2 pandemic and seasonal viruses revealed that PB2, PB1 and PA were responsible for the difference in polymerase activity between them. Five residues, one in PB2, three in PB1 and one in PA were identified to be significant for the polymerase activity change. These polymerase subunits and residues may act as part of the determining factors for the H2N2 pandemic virus.
Furthermore, PB2-627 has been shown to have stringent host specificity and affect polymerase activity and viral replication. Recombinant viruses in mammalian and avian cells with random mutation were generated at this position. It showed that the amino acids at this position are not restricted to those appear in the nature for generating viable viruses. It was also observed that the avian-derived viruses generally had lower polymerase activity and reduced growth kinetics in mammalian cells, while part of the mammalian-derived viruses had lower polymerase activity and reduced growth kinetics in avian cells. This consolidated the role of PB2-627 on host specificity and demonstrated the possibility of some novel amino acids for this position, which may play a role in the future influenza pandemic.
The 2009 H1N1 pandemic virus contains a reassorted vRNP with subunits of avian, human and swine origins. This prompts me to compare the polymerase activity of all the 81 possible combinations of chimeric vRNPs of three different origins. The results were statistically analyzed and several single subunit factors and interactions between vRNP subunits were identified to significantly affect the polymerase activity. In order to reduce the effort and resources required, a fractional factorial design of 27 experimental runs was developed to substitute the 81-combination full factorial design for identifying the significant single subunit factors that affect the polymerase activity. Overall, this study identified some factors that may contribute to a pandemic virus and allows us to have better understanding of the role of polymerase in a pandemic virus. These findings may contribute to evaluating the pandemic potential of the novel virus that emerges or may emerge in the nature and enhances the preparedness towards the next pandemic influenza.published_or_final_version
Public Health
Doctoral
Doctor of Philosophy
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GREEN, GEORGE DAVID. "QUINODIMETHANE POLYMERS." Diss., The University of Arizona, 1986. http://hdl.handle.net/10150/183860.
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Monomers and polymers containing the quinodimethane unit were synthesized. The infrared, nuclear magnetic resonance (NMR) and ultraviolet spectra of these materials were also reported. Oxidation of the enolate dianoion of α, α'-di(carbomethoxy)-α, α'-diphenylquinodimethane (DMPQH₂) with iodine gave 7,8-di(carbomethoxy)-7,8-diphenylguinodimethane (DMPQ) in greater than 50% yield. This compound had a reduction potential of -0.85V (Ag/AgCl reference) but would not form charge transfer complexes with electron donors. Attempted polymerization reactions of DMPQ were also discussed. A series of donor - acceptor substituted quinodimethanes was synthesized and a discussion of their electronic properties was included. Condensation copolyesterification of two of these materials was performed. Polymers with inherent viscosities ranging from 0.11 dL/g to 0.45 dL/g were obtained. The polymers were soluble at room temperature in hexafluoroisopropanol (HFIP) and a 1 : 1 solution of phenol/chlorobenzene. The polymers were also soluble at elevated temperatures in dipolar aprotic solvents.
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Dann, A. J. "Phthalocyanine polymers." Thesis, University of Nottingham, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376167.
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Smith, A. E. "Functionalised polymers." Thesis, Aston University, 1988. http://publications.aston.ac.uk/9733/.
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Synthetic routes to polymers possessing functional groups were studied. Direct functionalisation of poly(vinyltoluene) by lithiation and carboxylation resulted in the expected carboxylic acid but reaction was complicated by the production of a mixture of products. Reaction occurred both at the polymer backbone and at the pendant methyl group. Reaction with ethyl formate was also difficult to control and a secondary alcohol was formed even when an excess of the carbonyl compounds was employed. Grignard formation of poly(bromostyrene) was successful but once formed, the derivative rearranged resulting in chain scission and degradation of the polymer. Therefore subsequent reactions of the Grignard reagent with carbonyl groups were unsuccessful in producing functionalised polymers. Reactions of vinyltoluene monomer were more successful. Although complications arose when lithiation and carboxylation of the monomer were carried out using lithium diisopropylamide because the carboxylic acid product reacted with the excess lithium diisopropylamide present, metallation by potassium t-butoxide followed by reaction with 2-(3-chloropropyl)-2-methyl-1,3-dioxalane resulted in the formation of 2-methyl-2(4-(vinylphenyl)-butyl-1,3,-dioxalane. The butyllithium initiated anionic polymerisation of this protected monomer resulted in a polymer which had a very narrow molecular weight distribution (Mw/Mn= 1.05) and subsequent hydrolysis of the polymer resulted in poly(6(vinylphenyl)-hexan-2-one) which was derivatised with 2,4 dinitrophenyl-hydrazine. Functionalisation by modification of the siloxane derivative 3-(methylpropenoxycarbonyl)ltrimethoxysilane was unsuccessful. The acid catalysed exchange reactions of this monomer with alcohols such as eugenol, octan-1-ol, pentan-1-ol, and hexan-1-ol were inefficient, resulting in a mixture of products and unreacted starting materials.
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Arndt, Joseph W. "Characterization and structural determination of metalloenzymes DNA polymerase beta, carboxypeptidase, and acetyl coenzyme-A decarbonylase/synthase /." Columbus, OH : Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1061312369.
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Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xxii, 172 p. : ill., some col. Includes abstract and vita. Advisor: Michael K. Chan, Dept. of Chemistry. Includes bibliographical references (p. 165-172).
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Liu, Karen Ka Yan. "Origins of shear strength of polymers and reinforced polymers." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ27996.pdf.
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Lai, Benjamin Fook Lun. "Bioactive polymers : a comparative study on the antithrombotic properties of soluble polymers and surface grafted polymers." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/20871.
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Use of synthetic materials in medical applications is one of the most common practices in modern medicine. Yet occurrence of surface-induced thrombus formation on these materials, especially those associated with cardiovascular applications, generates a need for surface modifications. Limiting thrombus formation on a biomaterial surface represents the ultimate success for blood contacting devices. One interesting approach is to enhance fibrinolysis before the blood clot becomes stabilized. Herein, two synthetic polymers, poly-N- [(2, 2-dimethyl-1, 2-dioxolane) methyl] acrylamide (PDMDOMA) and poly- (N-isopropylacrylamide) (PNIPAm), were tested for this particular antithrombotic property. Surface-grafted PNIPAm samples, brush-PNIPAm and star-PNIPAm, were also tested for the biological activity.
We evaluated the influence of these synthetic polymers on blood hemostasis by studying the fibrin polymerization process, the three-dimensional clot structure, and the mechanical properties of blood clot such as its clot strength, clot elasticity and clot fibrinolysis. Both linear PDMDOMA and PNIPAm altered the normal fibrin polymerization by changing the rate of protofibril aggregation and resulting in a 5-fold increase in the overall turbidity. Fibrin clots formed in presence of these synthetic polymers exhibited thinner fibers with less branching and resulted in a more porous and heterogeneous clot structure in scanning electron micrographs. The structural changes in these clots led to significant difference to their mechanical properties. Lower clot strength and clot elasticity were recorded from the thromboelastography study. More interestingly, enhanced clot lysis was measured by thromboelastography when whole blood was clotted in presence of PDMDOMA or PNIPAm. Further evidence of the altered clot structure and clot cross-linking was obtained from the significant decrease in D-dimer levels measured from degraded plasma clot. Similar results were obtained when star-form of PNIPAm was used but not for brush-form PNIPAm.
The antithrombotic activity of soluble PDMDOMA and PNIPAm could potentially lead to the development of novel antithrombotic agents that could enhance endogenous fibrinolytic activity by modulating the fibrin clot structure. In the exploratory analysis of surface grafted PNIPAm (brush-PINPAm), brush-PNIPAm showed that the biological activity of attached chains is quite different from soluble polymers and several parameters need to be optimized to generate an antithrombotic coating for biomaterials.
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KUO, THAU-MING. "SYNTHESIS AND CHARACTERIZATION OF MONOMERS AND POLYMERS CONTAINING MULTIPLE P-ARYLENEAZO OR P-BENZOQUINODIIMINE GROUPS: CONDUCTING POLYMERS, LIQUID CRYSTAL POLYMERS, AND DIPOLAR POLYMERS." Diss., The University of Arizona, 1987. http://hdl.handle.net/10150/184215.
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Aniline Black, a polymer containing p-benzoquinodiimine groups, was synthesized chemically or electrochemically by the oxidation of aniline. The polymer salts showed the conductivity of 10⁻¹-10⁻³ ohm⁻¹cm⁻¹. Polymers containing anthroquinodiimine units were also prepared by polycondensations. The syntheses of model compounds containing p-benzoquinodiimine were attempted. Multiazobisphenol monomers were synthesized. 4,4'-(3,3'-Dimethyl-4,4'-biphenylenebisazo) bisphenol 7, 4,4'-[azobis(p-phenyleneazo)] bisphenol 8, and 4,4'-(2-methoxy-1,4-phenylenebisazo) bisphenol 10 displayed liquid crystal (l.c.) properties, while model derivatives of 7, 8, and 4,4'-(4,4'-stilbenebisazo) bisphenol 9 did likewise. Monomers and derivatives of 4-[(4-hydroxyphenyl)azo]-1-naphthol 5, and 4,4'- [oxybis(p-phenyleneazo)] bisphenol 6 showed no l.c. behavior. New thermotropic polyesters based on these multiazobisphenols were synthesized. Sebacates of 5, 6, 7, 8, and 10 showed l.c. behavior, while polymers based on isophthalic or 5-t-butylisophthalic acid did not do so. Polyformals were also synthesized from these momoners, only that of 4 showed weak l.c. behavior. The correlation between the structure of these polymers and their tractabilities, electrical properties, liquid crystal behaviors was studied. Polymers and copolymers containing p-azoarylene and p-azoxyarylene groups were synthesized by oxidative coupling of various aromatic diamines. Films were cast directly from the reaction mixtures or from the polymer solution. The films were n-doped by sodium naphthalide or p-doped by iodine. They showed electrical conductivities of 10⁻⁴ to 10⁻⁵ ohm⁻¹cm⁻¹. AB monomers containing dipolar p-phenyleneazo groups were synthesized: 4-(4-hydroxy-2-methoxyphenylazo) benzoic acid 21, 4-[4-(4-hydroxy-2-methoxyphenylazo)-2-methoxyphenylazo] benzoic acid 22, and 4-(4-hydroxy-2-methoxyphenylazo)-3-nitrobenzoic acid 23. The monomers were polymerized by direct polycondensations. The polyester synthesized from 21 formed a red, transparent film. A polymethacrylate containing dipolar p-phenyleneazo groups in the side chains was also prepared by the free radical polymerization of 1- [3-methoxy-4-(p-nitrophenylazo)-phenoxy] hexyl methacrylate 28.
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Mäkiniemi, M. (Minna). "Human DNA polymerase ε associated proteins:identification and characterization of the B-subunit of DNA polymerase ε and TopBP1." Doctoral thesis, University of Oulu, 2001. http://urn.fi/urn:isbn:9514259688.
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Abstract
DNA polymerase ε from HeLa cells has been purified as a heterodimer of a 261 kDa catalytic subunit and a tightly associated smaller polypeptide, the B-subunit. The cDNAs encoding the B-subunits of both human and mouse Pol ε were cloned and shown to encode proteins with a predicted molecular weight of 59 kDa. These subunits are 90 % identical and share 22 % identity with the 80 kDa B-subunit of Saccharomyces cerevisiae Pol ε. The gene for the human Pol ε B-subunit was localized to chromosome 14q21-q22 by fluorescence in situ hybridization.
Primary structure analysis of the Pol ε B-subunits demonstrated that they are similar to the B-subunits of Pol α, Pol δ and archaeal DNA polymerases, and comprise a novel protein family of DNA polymerase associated-B-subunits. The family members have 12 conserved motifs distributed in the C-terminal parts, which apparently form crucial structural and functional sites. Secondary structure predictions indicate that the B-subunits share a similar fold, and phylogenetic analysis demonstrated that the B-subunits of Pol α and ε form one subfamily, while the B-subunits of Pol δ and the archaeal proteins form a second subfamily. The corresponding eukaryotic and archaeal catalytic subunits are not related, but all have the characteristics of replicative DNA polymerases. This indicates that the B-subunits of replicative DNA polymerases from archaea to eukaryotes belong to the same protein family and perform similar functions.
In S. cerevisiae, Pol ε associates with the checkpoint protein Dpb11. In this study, a human protein, TopBP1, with structural similarity to the budding yeast Dpb11, fission yeast Cut5 and the breast cancer susceptibility gene product Brca1 was identified. The human TOPBP1 gene localizes to chromosome 3q21-q23 and encodes a phosphoprotein of 180 kDa. TopBP1 has eight BRCT domains and is also closely related to the recently identified Drosophila melanogaster Mus101. TopBP1 expression is induced at the G1/S boundary and it performs an important role in DNA replication, as evidenced by inhibition of DNA synthesis by TopBP1 antiserum in isolated nuclei. TopBP1 also associates with Pol ε and localizes, together with Brca1 to distinct foci in S-phase, but not to sites of ongoing DNA replication. Inhibition of DNA replication leads to re-localization of TopBP1 and Brca1 to stalled replication forks. DNA damage induces formation of distinct TopBP1 foci that co-localize with Brca1 in S-phase, but not in G1-phase. The role of TopBP1 in the DNA damage response is also supported by the interaction between TopBP1 and the human checkpoint protein hRad9. These results implicate TopBP1 in replication and checkpoint functions.
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Thuresson, Ann-Charlotte. "Regulation of Mammalian Poly(A) Polymerase Activity." Doctoral thesis, Uppsala University, Department of Cell and Molecular Biology, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-2892.
Full textAbstract:
Poly(A) polymerase (PAP) is the enzyme catalyzing the synthesis of the adenine tail to the 3’-end of mRNA. This A-tail is present on the majority of the primary RNA transcripts of protein-coding genes, and is important for mRNA stability, export to the cytoplasm and translation. Therefore, PAP is a key regulator of eukaryotic gene expression. This thesis describes the heterogeneity of PAP and the functional significance of multiple isoforms of PAP.
PAP exists in many different isoforms generated by three different mechanisms, gene duplication, alternative mRNA processing and post-translational modification. In HeLa cell extracts three different forms of PAP being 90, 100 and 106 kDa in size have been detected, where the 106 kDa isoform is a phosphorylated version of the 100 kDa species. It is shown that the N-terminal region of PAP contains a region required for catalysis, while the C-terminal end is important for the interaction with the cleavage and polyadenylation specificity factor (CPSF). Interestingly, it was found that also the extreme N-terminal end is important for the interaction with CPSF. This region is post-translationally modified by phosphorylation. Five alternatively spliced forms of PAP mRNAs are encoded by the PAPOLA gene while one unique species is encoded by the PAPOLG gene. The analysis showed that the exact structure of the alternatively spliced C-terminal end of PAP played an important role for catalytic efficiency. Thus, the C-terminal end contains a region important for modulating the catalytic efficiency of PAP.
Aminoglycoside antibiotics inhibit PAP activity, most likely by displacement of catalytically important divalent metal ions. Data shows that different aminoglycosides inhibit PAP activity by different mechanisms suggesting that the binding sites for the different aminoglycosides do not completely overlap. It is concluded that aminoglycosides interfere with enzymes important for housekeeping functions in mammalian cell, which may explain some of the toxic side effects caused by aminoglycoside antibiotics in clinical practice.